Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish. In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot’s smooth muscle relaxation. Together, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

中文翻译：

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双壳类动物没有什么不同：一氧化氮是太平洋牡蛎变态的负调节因子，巨牡蛎


一氧化氮 (NO) 被认为是许多无脊椎动物物种变态的调节因子，尽管 NO 途径在腹足类、环节动物和甲壳类动物中已得到相对充分的研究，但关于 NO 对双壳类变态影响的研究非常有限贝类。在本文中，我们研究了 NO 途径抑制剂和 NO 供体对太平洋牡蛎 (Crassostrea gigas) 幼虫变态诱导的影响。一氧化氮合酶 (NOS) 抑制剂 s-甲基异硫脲半硫酸盐 (SMIS)、氨基胍半硫酸盐 (AGH) 和 7-硝基吲唑 (7-NI) 分别在 75%、76% 和 83% 时诱导变态，并且以浓度依赖性方式起作用方式。暴露于 1H-[1,2,4]恶二唑[4,3-a]喹喔啉-1-酮 (ODQ) 导致高达 54% 的额外诱导，这是一种可溶性鸟苷酸环化酶抑制剂，NO 与该酶相互作用以催化环单磷酸鸟苷（cGMP）的合成。相反，高浓度的NO供体硝普钠二水合物与变态诱导剂肾上腺素、MK-801或SMIS相结合，显着减少变态，尽管不能排除与变态途径无关的过量NO的潜在有害影响。无论使用何种诱导剂，变态后幼虫中 CgNOS 的表达也会降低，但在幼苗中变态后再次增强。用 DAF-FM 二乙酸盐荧光检测感受态幼虫中的 NO，并通过原位杂交定位牡蛎一氧化氮合酶 CgNOS 表达，结果表明 NO 主要出现在两个关键的幼虫结构中，即帆和足。通过免疫荧光检测，cGMP 也在足部被检测到，并且可能与足部平滑肌松弛有关。 总之，这些结果表明，NO 途径作为太平洋牡蛎幼虫变态的负调节因子，并且 NO 的减少通过抑制游泳或爬行行为以及一系列额外的神经内分泌下游反应来诱导变态。